ISOLATION AND CHARACTERIZATION OF RAT-LIVER AMPHISOMES - EVIDENCE FORFUSION OF AUTOPHAGOSOMES WITH BOTH EARLY AND LATE ENDOSOMES

Amphisomes, the autophagic vacuoles (AVs) formed upon fusion between a utophagosomes and endosomes, have so far only been characterized in in direct, functional terms. To enable a physical distinction between aut ophagosomes and amphisomes, the latter were selectively density-shifte d in sucrose gradients following fusion with AOM-gold-loaded endosomes (endosomes made dense by asialoorosomucoid-conjugated gold particles, endocytosed by isolated rat hepatocytes prier to subcellular fraction ation). Whereas amphisomes, by this criterion, accounted for only a mi nor fraction of the AVs in control hepatocytes, treatment of the cells with leupeptin (an inhibitor of lysosomal protein degradation) caused an accumulation of amphisomes to about one-half of the AV population. A quantitative electron microscopic study confirmed that leupeptin in duced a severalfold increase in the number of hepatocytic amphi somes (recognized by their gold particle contents; otherwise, their ultrastr ucture was quite similar to autophagosomes), Leupeptin caused, further more, a selective retention of endocytosed AOM-gold in the amphisomes at the expense of the lysosomes, consistent with an inhibition of amph isome-lysosome fusion. The electron micrographs suggested that autopha gosomes could undergo multiple independent fusions, with multivesicula r (late) endosomes to form amphisomes and with small lysosomes to form large autolysosomes. A biochemical comparison between autophagosomes and amphisomes, purified by a novel procedure, showed that the amphiso mes were enriched in early endosome markers (the asialoglycoprotein re ceptor and the early endosome-associated protein I) as well as in a la te endosome marker (the cation-independent mannose 6-phosphate recepto r). Amphisomes would thus seem to be capable of receiving inputs both from early and late endosomes.